Problems: Retroviral Gene Transfer

[Hans Bojar]

Hello 
				
Does anyone have experience in generating high level retroviruses 
for retroviral gene transfer? We want to use the PA317 retroviral packaging 
line with the pLXSN retroviral vector. But we have some theoretical and 
practical problems: 

1.)    Why produce a transfected helper line a lower virus titer
       than a second helper line, which has been infected by a 
       transfected first?        (esp. PA317 --> PE501)

2.)    Does a transfected retroviral vector integrate into the 
       genome of the helper cell line? If not, how is the vector
       replicated in a growing cell line under neo-selection?
       ( we could not detect an eucariotic oriR in pLXSN ).

3.)    In order to generate a stable virus producing cell line, we need the 
       cell line PE501, the ecotropic derivat of PA317 (ATCC 9078), but we 
       could not find a soure for PE501.       

******   Every answer helps